Copper-containing proteins function in a variety of biological oxidations, some of which are involved in the metabolism of certain vitamins and hormones as well as a host of other systems. Three fundamental types of copper chromophores are found in proteins, called type 1 or blue copper, type 2 and type 3, respectively. The role of blue copper is electron transport, whereas the latter two types appear to interact with substrates as well as to undergo valence changes. In particular the type 3 site interacts with the dioxygen molecule. Recently this investigator prepared the Co(II) derivatives of several blue copper proteins and initiated physical studies of these derivatives which have revealed some aspects of the electronic structures and coordination geometry of blue copper. We now propose to initiate H1-nmr studies of several blue copper proteins and their Co(II) derivatives in order to determine the binding groups at the blue site. In addition we propose to extend the Co(II) replacement studies to a blue site with a higher reduction potential and to prepare an artificial blue protein. Studies of the latter system will allow us to probe directly the properties of a Cu(II)-sulphydryl chromophore. Other important studies proposed are chemical and physical studies of the type 3 site, including (1) epr studies of the partially reduced type 3 site, (2) ligand binding studies of the reduced type 3 site and (3) H1-nmr studies of a copper-containing oxidase. Important goals of the research proposed herein are (1) to determine the chemical basis for the large variation in reduction potentials for blue copper sites - the nature of the ligand groups and the coordination geometry, (2) to determine the structural nature of the type 3 site and (3) to probe the intersite interactions which occur in the oxidase enzymes. These interactions are an important feature of the mechansim by which these essential enzymes mediate the four electron reduction of dioxygen which is the fundamental concern of all aerobic organisms.